Targeting mechanisms that are especially important in the DNA damage response of pre-malignant and cancer cells may lead to development of novel chemosensitizing agents. Cancer cells are deficient in the primary G1/S cell cycle checkpoint, often through p53 functional loss. Therefore, their survival after DNA damage is more dependent on later cell cycle checkpoints such as the replication checkpoint that ensures DNA is copied fully before mitosis begins. Caffeine is the most commonly used inhibitor of this checkpoint and our prior studies have shown promise for caffeine as an inhibitor of ultraviolet-induced skin cancer by suppressing the ATR/Chk1 pathway required for replication checkpoint function. Beyond the unique case of UV-induced skin malignancies, caffeine cannot be used to sensitize cancers due to non-specific toxicity at the required doses. Through an unbiased screen for DNA damage response inhibitors, we discovered four novel compounds that are non-toxic to cells when given alone, but sensitize p53-deficient cells to diverse classes of chemotherapeutic agents. All four compounds disrupted the normal cellular response to DNA stresses, inhibited Chk1 phosphorylation via mechanism(s) that are distinct from that of caffeine, and were >250 times more potent than caffeine. The broad long-term goals of this proposal are: to gain insight into therapeutically targetable components of the DNA damage response; to explore the feasibility of using these targets to make cancers more sensitive to existing chemotherapeutics; and to further examine the unique potential of using checkpoint inhibition to reduce UV-induced skin cancer. In Aim 1 we will determine the general mechanistic class to which the novel compounds belong as inhibitors of Chk1 phosphorylation and hypothesize that studying these inhibitors isolated through an unbiased chemical genetic screen will provide new mechanistic insights on the DNA damage response. In Aim 2 we will test the hypothesis that the identified compounds will act as chemosensitizers in vitro and in vivo when combined with DNA-damaging agents. In Aim 3 we will test the hypothesis that checkpoint inhibition will be therapeutically useful to eliminate UV-damaged cells and prevent UV-induced skin cancers. We thus propose to extend our work from the prior funding period to further develop the rational biological basis and practical means to selectively target malignant cells to established therapies.